It is our goal to continue our molecular and genetic analysis of synaptic transmission in the soil nematode Caenorhabditis elegans. We have identified a set of genes which appear to encode presynaptic proteins; these genes, when mutated, cause accumulation of excess acetylcholine and also confer resistance to inhibitors of acetylcholinesterase. Some of these genes have been shown to encode important synaptic vesicle proteins such as synaptotagmin and the vesicular acetylcholine transporter. Another gene, called unc-41 has recently been cloned and appears to encode a novel presynaptic protein. The underlying theme of this research is the identification of new presynaptic proteins and the determination of their function. The three broad goals of the proposed research are: (l) to identify new genes affecting presynaptic function through genetic and phenotypic analysis of a collection of mutants resistant to acetylcholinesterase inhibitors; (2) continue the molecular analysis of the unc-41 gene and the UNC-41 protein; and (3) begin detailed molecular studies on the newly identified presynaptic genes. C. elegans is being used for these studies because of its simple nervous system, its ease of genetic and molecular analysis, and the availability of the technique of DNA-mediated transformation to introduce cloned genes into this organism. The studies on unc-41 are aimed at understanding the role of the UNC-41 protein in synaptic transmission. The information obtained about wild-type gene structure and transcripts will be used to analyze a collection of unc-41 mutants. Antibodies directed against the UNC-41 protein will be produced and used for immunolocalization studies. Mutations will be isolated in genes which interact with unc-41; such mutations might encode additional presynaptic components. The investigations of unc-41 will provide a paradigm for the analysis of additional presynaptic genes. Although this is basic research, it is clearly relevant to health, since proper neurotransmitter release is crucial to proper function of the nervous system, and defective transmitter release has been implicated in many psychiatric and neurological disorders.